A LABORATORY MANUAL A LABORATORY MANUAL 
OF PHYSIOLOGICAL AND 
CLINICAL CHEMISTRY 
AND TOXICOLOGY 
By ARTHUR E. AUSTIN, A.B., M.D. 
Professor of Medical Chemistry, Medical Department of 
Tufts University, Boston 
AND 
Laboratory Assistant in Medical Chemistry, Medical 
Department of Tufts University, Boston 
ISADOR H. CORIAT 
LAMSON, WOLFFE AND COMPANY 
Boston. New York. London Mdcccxcviii Copyright, 1S98 
By Lamson, Wolft'e and Company 
All rights resented 
PRESS or 
$otM»ell anti Cjjurcfjtll 
BOSTON Table of Contents. 
Preface ix 
Page 
PART I. 
Physiological Chemistry 
I. Egg Albumen 3 
IT. Nucleo-Proteids 6 
Nucleo-Album e n 8 
III. Albuminoides 9 
IV. Blood-Serum 10 
V. Albumoses 12 
VI. Sugars and Starches . . . 13, 10 
VII. Fats, Fatty Acids, and Glyc- 
erine 17, 19, 20 
VIII. Chemistry of Blood 21 
IX. Clinical Analysis of Blood ... 26 
X. Saliva 32 
XI. Chemistry of Gastric Juice' . . 33 
XII. Clinical Analysis of the Stomach 
Contents 37 
XIII. Bile 41 
XIV. Pancreatic Juice 43 
v vi 
Contents 
PART II. 
The Urine. 
Page 
I. General Characteristics .... 49 
II. General Chemistry and Clinical 
Analysis 50 
III. Sediment 60 
PART III. 
Toxicology. 
I. Acids: 
Sulphuric . . 67 
Nitric 67 
Hydrochloric 68 
Oxalic ,69 
Carbolic .69 
Hydrocyanic 70 
II. Metallic Poisons: 
Arsenic .71 
Antimony 72 
Mercury 73 
'ill. Metallic Poisons (continued) : 
Copper 74 
Lead 75 Contents 
vii 
Phosphorus 76 
Nitro-Benzol 76 
Page 
IV. Alkaloids: 
Morphine 78 
Strychnine 78 
Veratrine 79 
Atropine 79 
Cocaine 80 
Stas-Otto Method 80 
V. Scheme for the Detection of an Un- 
known Poison 82 
Appendix. 
Reagents 87 
Standard Solutions . . . . . . . 92 
Scheme for Analysis of Blood .... 95 
Scheme for Analysis of Gastric Contents . 96 
Scheme for Analysis of Urine . . . . 97 Preface. 
To offer to the medical profession in general 
and to the medical student in particular a 
new work embodying laboratory methods of 
clinical chemistry and toxicology, where so 
many admirable treatises are in existence, may 
appear at first sight an unprofitable and 
useless labor. But all such previous works 
have either been too voluminous for general 
use, or too complicated to serve as an ordinary 
working; manual. What the student needs 
is a work brief enough to give him a clear 
understanding of the methods of chemical 
diagnosis, and broad enough so that its pages 
may comprise all that he will need in the 
intelligent practice of his profession. While 
this little work does not pretend to be a com- 
plete working manual of medical chemistry 
and toxicology, if it accomplishes the purpose 
for which it was written, that of leading the 
ix X 
Preface 
student by simple steps to the more compli- 
cated methods of clinical chemical diagnosis, 
the authors will be thankful that their labors 
have not been in vain. Our thanks are due 
to Dr. Thorpe for his many useful suggestions 
and kind assistance while in preparation of 
this manual. 
A. E. Austin. 
I. H. Corn at. 
Boston, July, 1898. PART I. 
PHYSIOLOGICAL CHEMISTRY. L 
Egg Albumen. 
Take 20 c.c. egg albumen (white of egg) 
and shake well in a flask with 150 c.c. of 
water. Filter, and the flltrate, apart from a 
slight opalescence, should be wholly clear. 
With the filtered fluid try the following tests : 
1. Heat a portion in a test-tube to boiling, 
and there is a white cloudiness, but a separa- 
tion of coagulated albumen does not take place. 
This follows by the careful addition of dilute 
acetic acid (10%), drop by drop. The reac- 
tion of all albumens must be acid for coagu- 
lation. 
2. To a portion in a test-tube add one-half 
the amount of concentrated salt solution and 
divide into two parts. Heat one part to boil- 
ing by itself, and there occurs a flocculent pp, 
or coagulation. To the other part add acetic 
acid until slightly acid, and it becomes cloudy 
in the cold, but gives on heating a flocculent 
pp. The greater amount of salt an albumen 
solution contains, so much more independent 
of the acidity is the coagulation by heating. 4 
A Laboratory Manual 
3. To a portion in a test-tube add glacial 
acetic acid, and heat. Acid albumen is formed. 
Now cool the tube and add NaOH, and a pp. is 
formed, while the reaction is still slightly 
acid; but if NaOH be added in excess, the 
pp. is dissolved and alkali albumen is formed. 
4. Heat a portion with half the amount of 
NaOH, and there is formed alkali albumen. 
Cool and add dilute II.2S04— the albumen is 
pp., but again dissolved in an excess of the re- 
agent. 
5. To a portion in a test-tube add CuS04, 
and there arises a bluish-white pp. of copper 
albuminate, which, on the addition of NaOH, 
is dissolved in a deep-blue fluid. The salts cf 
the other heavy metals in general give pps. 
6. To a portion in a test-tube add HgCl2. 
There arises a thick, white pp. which is insol- 
uble in excess of the reagent, but soluble in 
cone, salt solution. 
7. Heat a sample with strong NaOH and 3 
drops of neutral lead acetate. The solution is 
blackened, due to the separation of sulphur. 
Upon acidifying with HC1, there is a separa- 
tion of large black flakes, while in serum albu- 
men there is only a cloudy gray fluid. This 
depends upon the fact that the sulphur in egg 
albumen is separated in larger quantities, and Of Physiological Chemistry 
5 
the lead albuminate is less soluble in HC1 than 
that of serum albumen. 
8. Shake a portion with ether, and there is a 
complete coagulation. 
9. To a small portion add cone. HN03, and 
there arises a pp. which is not dissolved even 
when the amount of acid equals half the amount 
of albumen solution. To this end it is neces- 
sary to add much larger quantities or to warm. 
Color Tests for Albumen.' 
10. Biuret Test. — To a portion in a test- 
tube add an equal amount of NaOH, and 
then a few drops of a very dilute solution 
CuS04. (One drop CuS04 to a test-tube of 
water.) Purple to amethyst color. 
11. Millon’s Test. — To a portion add a few 
drops of Millon’s reagent, and boil. Brick-red 
pp. 
12. Adamkiewicz Test.— Take one part 
cone. II2S04 and two parts of acetic acid. Add 
the albumen solution carefully without shaking, 
so that it shall overlay the acids. Warm 
slightly, and at the point of contact there 
develops a beautiful purple-red zone, which 
grows more intense in standing. 
13. Xantlio Proteic Reaction. — To a por- 
tion add strong HN03 until the pp. which is 6 
A Laboratory Manual 
formed no longer dissolves. Heat, and the pp. 
becomes colored yelloAA\ Noav cool and add 
NaOH in excess — color changes to orange. 
Now take 1 part of the albumen solution 
and nine of water and perform all of these tests 
again. Note particularly the action with Cu 
(used in sugar tests) — with Hg (used in urea 
test)—acetic acid (used in mucus test) — 
and with Pb (action similar to that of bismuth, 
also used in sugar test). 
H. 
Nucleo-Proteids. 
Take £ cake of yeast, place in a mortar, add 
50 c.c. of water and 3 to 5 drops of a very 
dilute solution NaOH (until neutral or 
slightly alkaline). Rub well with a pestle, 
filter, and with the filtrate perform the fol- 
lowing; tests : 
© 
1. Heat a small portion to boiling in a test- 
tube. It should coagulate. How does it differ 
from egg albumen? 
2. Add to a portion, in a test-tube, dilute 
(10%) acetic acid. There arises a pp. which 
is soluble in excess of the reagent. 
3. Take 25 c.c. of the solution and add 
AgXOg until all the NaCl is precipitated. Filter, Of Physiological Chemistry 
7 
pass II2S through the filtrate until all the silver 
is precipitated as sulphide of silver. Warm 
and filter again (filter more than once if 
necessary, in order to get a clear filtrate). 
Take 10 c.c. of this solution and add an equal 
amount of dilute H2S04‘, and warm for some 
time (at least 20 minutes) upon the water-bath. 
Cool, alkalinize with NH4OH (there must be 
an excess), and filter. To the filtrate add 
silver nitrate — and there arises a white pp. of 
xanthine bodies. 
4. Take 10 c.c. of the solution, add 2 c.c. 
of cone. HC1, and cook for some time upon the 
water-bath, replacing the water as it evaporates. 
Then cool, neutralize with Nadi, add a few 
drops of CuSo4, and heat again to boiling. 
There arises a separation of red or yellow sub- 
oxide of copper, which is best seen upon plung- 
ing the tube into cold water. This is a 
reducing body, but it is probably not sugar. 
5. With a small portion try the biuret reac- 
tion. Purple to amethyst color. 
6. Try Milton’s reagent. Brick-red pp. 
7. Add alcohol to a portion and note the 
effect. Coagulation? 
8. Saturate a portion with MgS04 in sub- 
stance, warming slightly to effect complete 
solution. Note effect. Precipitation? 8 
A Laboratory Manual 
9. Take two yeast-cakes, make a solution of 
them in the same way as given above, and pre- 
cipitate the nucleo-proteid by warming and the 
careful addition of dilute acetic acid. Decant 
off the fluid, filter, and wash the pp. with water, 
alcohol, and ether. After thoroughly drying, 
rub up the pp. with 4 grammes saltpeter 
mixture,1 heat to melting in a crucible, dissolve 
the residue after cooling in nitric acid, and 
heat until the resulting nitrous acid fumes have 
been driven off. Take 10 c.c. of ammonium 
molybdate solution, and add to it the nitric 
acid solution drop by drop. A yellow pp. 
shows the presence of phosphorus in the form 
of the acid. To another portion of the solution 
add NH4OH in excess ; it should remain clear. 
Xow acidify slightly with acetic acid and add 
uranyl nitrate solution. There is formed a 
yellowish-white pp. if phosphoric acid is 
present. 
Nucleo- Albumen. 
Make a solution of casein (2 grammes of 
casein to 100 c.c. of water) and perform the 
same tests as for nucleo-proteids. Note 
whether nucleo-albumens contain — 
(1) Xanthine Bodies. 
1 This mixture is composed of 3 parts KNO3 and 1 part N‘a2C03. Of Physiological Chemistry 
9 
(2) A Reducing Body. 
(3) Phosphorus. 
m. 
Albuminoides. 
Take 5 grammes gelatin, add to it 100 c.c. 
water, and warm. It dissolves, but upon 
cooling it becomes semisolid, thus behaving 
directly opposite to albumen, which coagulates 
by heat. With the warm solution perform the 
following tests : 
1. To a portion add a few drops of Millon’s 
reagent, and boil. Brick-red pp. This is due 
to the gelatin containing albumen as an im- 
purity, as pure gelatin does not respond to 
Millon’s test. 
2. To another portion add an equal amount 
of XaOH and a few drops of weak copper 
sulphate solution. Biuret reaction. Amethyst 
color. 
3. Add nitric acid, and warm. Yellow color. 
Cool, and alkalinize with NaOH. Orange 
color. Xanthoproteic reaction. 
4. Cook a portion with NaOH and a few 
drops of subacetate of lead. Although gelatin IO 
A Laboratory Manual 
contains sulphur, there is no blackening, as the 
sulphur is less easily split off than in egg 
albumen. 
5. To a portion in 4 test-tubes add respec- 
tively : 
(a.) Nitric acid. No pp. 
(6.) Potassium ferrocyanide. No pp. 
(c.) Acetic acid. No pp. 
(d.) Copper sulphate. No pp. 
6. To a portion add some mercuric chloride. 
There is no pp. formed. Now add a few drops 
of HC1, and there is formed a pp. which is 
soluble in an excess of the acid. 
7. To a portion add some tannic acid. There 
is no pp. Now add some NaCl, and there is 
formed a copious pp. 
8. Try the Adamkiewicz reaction. A nega- 
tive result with pure gelatin. 
IV. 
Blood-Serum. 
Blood-serum, as well as serous exudations, 
contains an albumen soluble in water — the 
serum albumen; and one which is insoluble 
in water — the serum globulin. This latter is Of Physiological Chemistry 
11 
held in solution by the alkaline reaction and 
the salts of the serum. 
Rub 20 c.c. of blood-serum in a mortar with 
an excess of ammonium sulphate (about 15 
grammes) for a long time, in order that the 
fluid may be surely saturated with the salt. 
By this means both albumens are precipitated. 
Filter through a dry Alter, and the filtrate is 
albumen free. Heated to boiling and treated 
with acetic acid, it remains clear. Now wash 
the pp. with a half-saturated solution of ammo- 
nium sulphate, and the albumen in part becomes 
redissolved, while the globulin remaining upon 
the filter can easily be dissolved by water, with 
the aid of the salt mixed w7ith it. 
1. Solution of Globulin. — Add a few drops 
to half a test-tube of water. Precipitate. Solu- 
bility in water? 
2. Add a mere trace of very dilute NaOH 
to the previous pp. in the test-tube, and it 
becomes redissolved. 
3. Neutralize carefully with dilute HC1, and 
the globulin becomes again precipitated. If 
in (2) too much NaOII were added in pro- 
portion to the amount of globulin, it is not 
reprecipitated, for the NaCl formed holds it in 
solution. 
To these respective solutions, (a) serum 12 
A Laboratory Manual 
albumen and (b) serum globulin, add the 
reagents employed for egg albumen. Note 
how serum albumen and serum globulin differ 
from each other, and also how they both differ 
from egg albumen. 
OO 
Now take 10 e.c. of blood serum, add water 
to 100 e.c., and perform the following tests : 
1. Heat to boiling — no change. Add a 
drop or two of HN03 and heat again, and there 
is a separation of coagulated albumen. 
2. Add acetic acid and potassium ferro- 
cyanide. Cloudiness — then a pp. 
3. Acidify with HC1 and then add phospho- 
tungstic acid. Copious gelatinous pp. 
4. Add tannic acid. Copious pp. 
5. Add mercuric chloride. A pp. — which 
is soluble in NaCl solution. 
6. Add a few drops of Millon’s reagent, and 
boil. Reddish-brown pp. 
V. 
Albumoses. 
Use solution provided — 2 grammes to 100 
e.c. of water. 
1. Heat a portion to boiling; remains un- 
changed also after adding acetic acid (a drop) 
and NaCl. Of Physiological Chemistry 
*3 
2. Acidify with acetic acid and add cone, 
salt solution. The solution becomes cloudy, 
but upon heating again becomes clear. Upon 
cooling — cloudy. 
3. Add a few drops HN( )3, and there is a 
cloudiness to a pp. which is soluble in excess. 
By warming and NaOH — xanthoproteic re- 
action. 
4. Add a few drops acetic acid and the po- 
tassium ferrocyanide. Cloudiness, which dis- 
appears on heating (not always wholly). 
5. Biuret test — rose to purple color. 
(>. Perform the tests (3) — (4) — (5) — 
(6) — of blood-serum. Insoluble precipitates. 
VI. 
Sugars. 
1. Take a small portion of milk-sugar and 
heat it to melting upon platinum foil or a 
piece of tin plate. There is developed an odor 
of caramel and a carbonizing, with the final 
leaving of a slight amount of ash. 
Make a solution of 2 grammes milk-sugar to 
100 c.c. water, and perform the following 
tests: A Laboratory Manual 
H 
2. Trommer’s Test.— Add to a few c.c. of 
the solution an equal amount of NaOII, and, 
drop ‘ by drop, CuS04, shaking constantly. 
There arises a deep-blue solution, which upon 
heating gives a separation of red or yellow 
suboxide of copper. 
3. Moore’s Test.- Add to a portion an equal 
amount of NaOII and heat to boiling; yellow 
to brown color, with the odor of caramel, 
especially after acidification with dilute II2S04. 
4. Nylander’s Test. — Saturate a portion 
with sodium carbonate in substance, and boil. 
While boiling add a very small portion of bis- 
muth subnitrate and keep for some time at a 
boiling point. Gray to black color, with the 
separation of fine metallic bismuth. 
5. Add some ammonia and a few drops of 
AgN03, and heat. Separation of metallic 
silver in the form of a glistening mirror upon 
the test-tube. The mirror is especially marked 
if a drop of NaOH be added before boiling. 
As the AgNOg in this test is changed to 
fulminating silver, care must be taken against 
its possible explosion by afterwards filling the 
test-tube with water. 
6. Indigo Test.— Add indigo carmine to the 
sugar solution until distinctly blue, and then 
add sodium carbonate solution until alkaline, Of Physiological Chemistry 
15 
and warm. The solution becomes first violet, 
then red, yellow, and finally colorless. Pour 
half of the solution in another test-tube and 
shake with air. It again becomes blue, but 
upon heating is again decolorized. This con- 
tinues until all the sugar is used up by oxida- 
tion. 
7. Phenylhydrazin Test. — Take 25 c.c. of 
the solution in a small beaker, and add 1 
gramme phenylhydrazin hydrochlorate and 2 
grammes sodium acetate. Place in boiling 
water for 30 minutes, and then cool by plung- 
ing the beaker in cold water. A yellow 
crystalline deposit is thrown down, which, 
when examined under the microscope, is found 
to consist of wide prisms of phenyl-lactosazon. 
Now make a 2% solution of grape-sugar 
and perform the same tests. 
8. Fill two fermentation tubes with solutions 
of grape and milk sugar respectively. Shake 
with a piece of fresh yeast until disintegrated, 
and place in a brood oven until the next day. 
Note the difference. Milk-sugar ferments 
slowly and with difficulty. 
9. Grape-sugar can be dissolved in cone. 
HoSO without color; milk-sugar is colored 
brown when dissolved in the same acid. 
10. Phenylhydrazin Test. — Bright-yellow 16 
A Laboratory Manual 
needles of phenyl-glucosazon, arranged in 
bundles or sheaves. 
11. In two test-tubes place a few c.c. of 
grape and milk sugar respectively, and add 
one or two drops of a 15% alcoholic solution 
of alpha-naphthol. Then let about 1 c.c. of 
cone. H2S04 run carefully down the side of 
the test-tube so as to underlie the other fluid; 
there is formed at the junction of the two fluids 
a beautiful violet-red ring that increases inten- 
sity upon slight shaking. 
Make a 2 % solution of cane-sugar and try 
the reducing tests (copper and bismuth). Cane- 
sugar will not reduce. 
Try the fermentation test with yeast, the 
same as for grape and milk sugar. After fer- 
mentation again try the reducing tests. The 
cane-sugar is a disaccharide, and, by the action 
of the invertion of the yeast, has been split up 
into its two mono-saccharides, glucose and 
levulose, both of which will reduce. 
Cane-sugar does not form a compound with 
pheny lhydrazi n. 
Starches. 
Measure out 100 c.c. water, and with a 
portion of the same rub up 1 gramme of starch 
in a mortar. Pour into an evaporating dish Of Physiological Chemistry 
17 
and with the rest of the water wash all the 
starch into the dish. Now heat to boiling 
under constant stirring. Allow to cool and 
perform the following tests : 
1. Add to a portion in a test-tube 3 or 4 
drops of a solution of iodine and potassium 
iodide. Blue color. 
2. Roil a portion for 20 minutes with dilute 
HC1. Cool, and to a portion add iodine 
solution again. Mahogany-red color due to 
erythrodextrin. The starch has been converted 
to erythrodextrin and glucose (grape-sugar). 
Alkalinize another portion fully, and test for 
glucose with reducing tests. 
3. Take 10 c.c. of the starch solution, add 
1 c.c. saliva, and place in a brood oven until 
the next day. Test for erythrodextrin and 
maltose. 
VIL 
Fats. 
1. Rub a small portion of lard or oil upon 
paper (not filter-paper), and it becomes trans- 
lucent. 
2. Dissolve a piece of lard the size of a pea 
in a few c.c. of alcohol with the aid of a little 18 
A Laboratory Manual 
ether, and add a couple drops rosolic acid, 
which gives the solution a yellow color. Now 
add from a burette a A Normal sol. NaOH, 
and one or two drops changes the color to red. 
3. Rub up a small portion in a mortar with 
bisulphate of potash, and heat the mixture in a 
dry test-tube. Visible fumes and the pungent 
odor of acrolein show a neutral fat. 
4. Warm a small portion in a test-tube with 
sodium carbonate solution. The fat is dis- 
tributed in the solution, but the latter is not 
clear; saponification does not take place. 
5. Dissolve 5 grammes KOH in 35 c.c. alco- 
hol, by warming over the water bath. Then 
melt 5 grammes of lard over the water bath, 
and add to it while stirring the KOH solution. 
Cook for 20 to 30 minutes, or until a small 
portion in a test-tube of water shows no oil 
drops. There is complete saponification. 
C. Add to 65 c.c. water 20 c.c. dil. II2S04 
(20%) and warm somewhat, but not to boil- 
ing. Now add the soap solution, while still 
hot, carefully to the dilute acid (there is a 
liability to snap). Allow to cool, and the 
fatty acids arise to the surface of the fluid as a 
layer which can be broken through, and the 
watery solution which contains the glycerine 
can be poured off. Hot water should be added Of Physiological Chemistry 
*9 
to this fat, and the process repeated until the 
wash water is no longer acid. Dry the fatty 
acids between filter-paper. 
Fatty Acids. 
1. Behavior to paper same as neutral fat. 
2. Make a solution with alcohol and ether, 
and add NaOII as above (2), using rosolic 
acid as an indicator. It takes 2 or more c.c. 
to change the color to red. They are acid. 
3. No acrolein by heating with bisulphate of 
potash. 
4. Add to a portion a half-saturated solution 
of sodium carbonate, and heat. The fatty 
acids dissolve with the development of C02 
and the formation of a sodium soap. Dip the 
test-tube in water, and the soap solidifies. 
5. Dissolve 2 grammes of fatty acids in 100 
e.c. of water, with the addition of the smallest 
possible quantity of NaOII needed to effect solu- 
tion. With this solution perform the following 
tests : 
(«.) Add HC1. Separation of fatty acids. 
(b.) Addition of chloride of calcium. In- 
soluble chalk soap. The fluid no longer foams 
upon shaking. 
(c.) Add lead acetate, and warm. A white A Laboratory Manual 
20 
pp. which upon warming becomes tough and 
crumbly — " Lead Plaster.” 
(c7.) Take a little of the solution, add a few 
drops of oil, and shake. There is formed a flue 
white emulsion. 
(e.) Try the same thing with sodium car- 
bonate solution and oil. Negative result. An 
emulsion can only be formed when some free 
fatty acids are present, as when a fat becomes 
rancid. 
{/.) Place some of the fat in a test-tube 
and an equal amount of the fatty acids in 
another. Place both tubes in a beaker of 
water, and heat upon a piece of wire gauze, 
occasionally stirring the water so that the 
temperature shall be the same everywhere. 
It will be noticed that the fat melts much 
sooner than the fatty acids obtained from the 
same fat. 
Take some of the watery solution from Fats 
(6), and with it perform — 
1. Trommer’s Test. — Blue solution, but no 
reduction of copper. 
2. Mix some glycerine with a little borax 
and heat a small portion in the flame upon 
platinum foil. The flame is temporarily col- 
ored green. 
Glycerine. Of Physiological Chemistry 
2 I 
3. Try the acrolein test. 
4. Make an emulsion with oil and soap solu- 
tion, and examine under the microscope. 
VTIL 
Chemistry of Blood. 
1. Take a piece of lacmoid paper, wet with 
cone, salt solution, and place upon it with a 
glass rod a drop of blood. A blue spot shows 
the alkalinity of blood. 
2. Add to about 10 c.c. water a few drops 
of blood, shaking thoroughly. Then add some 
fresh tincture guaiac (made by dissolving a 
penknife-pointful of the powder with some 
alcohol in a test-tube) until the solution be- 
comes milky. Now add some old oil of tur- 
pentine, and upon shaking the solution becomes 
colored an intense blue. 
3. Add to a few c.c. blood several times its 
volume of peroxide of hydrogen. There arises 
a strong foaming and development of oxygen, 
with gradual decomposition of the blood-color- 
ing substance. This catalytic action of the 
blood is lost in the presence of hydrocyanic 
acid, but the acid must be added to the blood A Laboratory Manual 
22 
directly. Tn the blood of an animal killed by 
hydrocyanic acid, this catalytic action persists. 
4. Add to a little blood in a test-tube some 
ether and water, and shake thoroughly. The 
coloring matter of the corpuscles becomes dis- 
solved, and the blood becomes " lakey.” The 
biliary acids have the same action upon blood. 
5. Dilute 10 c.c. blood to 100 c.c. with 
water and perform the following tests: 
(a.) Take a half test-tube of blood, place 
in the spectroscope, and continue to add water 
until the two lines of oxyhaemoglobin in green 
and yellow can be seen. Now add a few drops 
of ammonium sulphide, and allow to stand for a 
few minutes. There is a change of color from 
red to green, and the appearance of only one 
line in the spectroscope, that of reduced haemo- 
globin, occupying a position midway between 
that of the other two. 
([b.) Take another portion of the blood solu- 
tion and add a few drops of freshly prepared 
cone, solution of potassium ferricyanide. The 
solution becomes brown and shows the charac- 
teristic four-line spectrum of methaemoglobin. 
Now add a few drops of ammonium sulphide, 
allow to stand for a short time, shake strongly, 
and again examine with a spectroscope. The 
spectrum of oxyhemoglobin will be found. Of Physiological Chemistry 
2 3 
Mettlernoglobin can be reduced to hemoglobin, 
and again oxydized to oxyhemoglobin. 
(c.) Pass hydrogen sulphide through some 
of the dilute blood and examine with a spectro- 
scope — the spectrum of sulph-hemoglobin. It 
resembles that of methemoglobin except that 
the line in red is farther to the left, and that it 
cannot be reduced. Try the reduction test, 
same as in (5). 
(d.) Add to 10 c.c. of the dilute blood 
1 c.c. NaOH, and heat. The solution be- 
comes brownish-green. Examination with the 
spectroscope shows one broad band of absorp- 
tion extending to red— the spectrum of lisema- 
tin in alkaline solution. Now add a few drops 
of ammonium sulphide, allow to stand for a short 
time, and again examine with a spectroscope. 
The one absorption band has disappeared, and 
in its place will be found the two lines of 
reduced haunatin. 
6. Pass ordinary illuminating gas through 
50 c.c. blood until it becomes a cherry-red. 
Examine with the spectroscope. The spectrum 
resembles that of oxyhemoglobin, except that 
it cannot be reduced. Try the reducing tests. 
(a.) Add to this blood and to genuine 
blood equal amounts (about \ its volume) of 
NaOH. Note the difference; place a drop of A Laboratory Manual 
24 
each in an evaporating dish, and note difference 
in color. 
(/>.) Add to 10 c.c. carbon monoxide 
blood 15 c.c. of a 20% solution potassium 
ferricyanide and 2 c.c. acetic acid — and to the 
same amount of unmodified blood add the same 
reagents. Allow to stand a few moments, and 
note the difference. 
(c.) To 5 c.c. of carbon monoxide blood 
add four times as much water. To a measured 
portion of this add three times its volume of a 
1 % tannic solution. Do the same with un- 
modified blood, and note the difference. 
7. Dry a drop or two of blood upon a micro- 
scope slide, and rub up the dried blood with 
the smallest possible quantity of sodium chlo- 
ride. Place a cover glass upon it, and add a 
couple drops of glacial acetic acid to the edge 
of the cover glass, allowing the acid to flow 
under the latter. Now warm upon the water 
bath until evaporated, add a drop or two more 
of the acid, and allow to cool. Examine 
with a microscope (or l" objective). 
Small dark-brown, rhombic crystals of hamiin 
chloride show the presence of blood. Stains 
upon linen or wood can be soaked out in a 
normal salt solution and tested in the same 
way. These crystals can be obtained from Of Physiological Chemistry 
25 
mere traces of blood, and their presence is con- 
clusive of the presence of blood in medico-legal 
examinations. 
8. Take 30 c.c. fresh blood, add 8 times as 
much water, and heat to boiling, with constant 
stirring. While boiling add carefully dilute 
(10%) acetic acid until slightly acid, and 
filter. The filtrate must be clear. Evaporate 
the filtrate to a small volume, and divide into 
two halves. With one, try Trommer’s Test 
for sugar. The other half is to be still further 
concentrated, and a drop allowed to evaporate 
upon an object glass, and examined with a 
microscope. Crystals of NaCl. 
The rest is to be evaporated to dryness in 
an evaporating dish, heated to redness, cooled, 
and dissolved in a little water. Examine for 
sodium chloride with AgN03 —for phosphates 
with ammonium molybdate. 
If, in the above, after heating to redness, a 
black residue remains, add a little saltpetre 
mixture, and heat to redness again. After 
cooling, dissolve the residue, which should be 
colorless, in nitric acid, evaporate off the 
excess of the acid, and test for phosphates 
and chlorides as above. 
9. Dry the pp. formed in (8), take 1 gramme 
of the dried pp. and rub thoroughly in a mortar 2 6 
A Laboratory Manual 
with 4 grammes saltpeter mixture. Heat to 
melting in a crucible for about 20 minutes, and 
after cooling dissolve the residue in dilute sul- 
phuric acid (8 parts by weight cone. H2S04-f- 3 
parts by weight of water). Filter, and to a 
portion of the filtrate, which should be clear, 
add some potassium sulphocyanide. A blood- 
red color shows iron. 
IX. 
Clinical Analysis of Blood. 
Specific Gravity (Roy’s method). — A num- 
ber of test-tubes are two-thirds filled with a 
mixture of glycerine and water in different 
proportions, so that the specific gravity in the 
tubes shall vary between 1030 and 1070. 
Blood is then drawn from the tip of the finger 
or the lobe of the ear into a capillary tube con- 
nected with an ordinary hypodermic syringe. 
A drop of blood is placed in each-tube, in 
which it will sink as long as the specific gravity 
of the glycerine mixture is lower than that of 
the blood, while it will remain suspended in a 
mixture the specific gravity of which is equiv- 
alent to its owm. 
Normal specific gravity— 1.04G — 1.067. Of Physiological Chemistry 
27 
Alkalinity (Ldwy’s method). —Five c.c. of 
defibrinated blood, accurately measured, are 
mixed in a small flask with 45 c.c. water; if 
not defibrinated, the blood is mixed with the 
same amount of a 0.25% solution ammonium 
oxalate. Titrate the solution with a 1/25 nor- 
mal solution of tartaric acid, until a drop 
placed upon a piece of lacmoid paper (which has 
been previously soaked in a cone, solution of 
magnesium sulphate) gives rise to a blue tinge. 
Calculation — 1 c.c. normal solution 
tartaric acid = .OOlfi grammes NaOH. 
Normal alkalinity in terms of sodium hydrate 
= 182 to 218 milligrammes for every 100 c.c. 
of blood. 
Quantitative Estimation of Haemoglobin 
(Fleischl’s method).—The instrument used is 
the haemometer of Fleischl, and the principle 
of the method depends upon the comparison of 
the color of the blood, diluted with water, with 
that of a g’lass wedge stained with a red pig- 
ment. The principal part of the instrument 
consists of two semicircular compartments, 
one receiving light from a plaster-of-Paris 
reflector through the glass wedge and called 
the red chamber, the other receiving light 
directly from the reflector and called the white 
chamber. 28 
A Laboratory Manual 
To use the instrument, the two compart- 
ments are partially tilled with water, and the 
required amount of blood is obtained by placing 
one end of the capillary pipette in contact with 
a drop of blood obtained from the tip of the 
finger or the lobe of the ear. The pipette is 
immersed in the white chamber and rotated 
between the fingers. By this method the blood 
passes into the water, and any traces of blood 
remaining in the pipette are carefully washed 
out. The two compartments are then com- 
pletely filled with water, and the glass wedge 
so adjusted, by means of the screw, that the 
color in the two chambers shall be the same. 
The number facing the notch in the scale aper- 
ture of the platform will then indicate the per- 
centage of haemoglobin, that of a healthy 
individual corresponding to 100. As the nor- 
mal amount of haemoglobin contained in 100 
grammes of blood is 13.7 grammes, the number 
100 on the scale of Fleischl’s instrument corre- 
sponds to 13.7 %. 
Calculate % according to equation : 
100: 13. 7:: reading on the scale : X 
The Enumeration of the Red Blood Cor- 
puscles (Thoma-Zeiss method). — The instru- 
ment used is the Thoma-Zeiss blood-counting 
apparatus. Of Physiological Chemistry 
29 
Blood is drawn from the tip of the finger or 
lobe of the ear into the capillary pipette, to the 
mark 1 or 0.5, according to the degree of dilu- 
tion desired. The blood is then diluted with 
Pacini’s fluid, which is drawn into the pipette, 
past the bulb, until the mark 101 is reached. 
If the blood had previously been drawn to the 
mark 0.5, it now has been diluted 200 times; 
if to the mark 1, it has been diluted 100 
times. The contents of the bulb are now 
thoroughly mixed by shaking. A drop of the 
mixture is placed in the counting-chamber, so 
that it shall completely fill it, and the cover- 
slip is adjusted. The slide is placed under the 
microscope, a Y' or i" objective being used. 
Now focus until the small squares come dis- 
tinctly into view. Each one of these small 
squares is m.m. long, m.m. wide, and 
yL m.m. deep ; its cubic contents are therefore 
ToVo cukic m.m. Count the corpuscles in at 
least 16 small squares and take the average, 
which will give the number of corpuscles in one 
small square, or in cubic m.m. One cubic 
m.m. of this diluted blood will then contain 
4,000 times the number contained in one small 
square, and one cubic m.m. of the undiluted 
blood will contain the product of this figure 
multiplied by the degree of the original dilution. 3° 
A Laboratory Manual 
The Enumeration of the Red Blood Cor- 
puscles by the Hsematokrit. — The graduated 
glass tube is completely filled, by suction, with 
blood from the finger or ear. The blunt point 
of the tube is then quickly covered with the 
finger, and the tube fixed in the frame of the 
centrifuge. This is rotated for two to three 
minutes, and the number of corpuscles which 
have been thrown to the bottom of the tube 
are read off*. Multiply the reading on the 
tube by 100,000, and the product is the num- 
ber of red corpuscles in one cubic in.m. 
The Enumeration of the White Blood Cor- 
puscles (Thoma-Zeiss method).—This enu- 
meration is done with the same apparatus as 
for the red corpuscles, except that a special 
diluting pipette is used. The diluting fluid 
is a 0.3% solution acetic acid colored with 
methyl-violet, which destroys the red cor- 
puscles and brings out the white very dis- 
tinctly. The pipette is tilled with blood to the 
mark 0.5 if we wish to dilute 20 times, or to 
1 if a dilution of 10 times is desired. Then 
fill the pipette to the mark 11 with the acetic 
acid solution. Place a drop in the Thoma- 
Zeiss cell, use a or y' objective, and focus 
as above. As the number of white corpuscles 
is smaller than the red, it is necessary to count Of Physiological Chemistry 
3i 
the number of corpuscles in a whole field; 
that is, in the area of one microscopic circle. 
Then determine the cubic contents of this mi- 
croscopic circle, and from this calculate the 
entire number in one cubic m.m. 
The cubic contents are determined as follows : 
Count the number of cross vertical lines in the 
field of the microscope, remembering that 
the distance between each two lines is m.m. 
This gives the diameter of the circle. Divide 
the diameter by 2, which gives the radius; 
square the radius, and multiply the square of 
the radius by 3.1416. The product is the area 
of the circle, and this area, multiplied by 0.1, 
which is the depth of the cell in a fraction of a 
millimeter, gives the cubic contents of the 
microscopic circle. The number of white cor- 
puscles counted in this single field is there- 
fore contained in the fraction of one cubic m.m. 
represented by the cubic contents of the field 
counted. From this calculate the number of 
white blood corpuscles in one cubic m.m. of 
diluted blood, and this, multiplied by the de- 
gree of dilution (10 or 20), will give the entire 
number in one cubic m.m. 
Formula for the determination of the cubic 
contents of the circle : 
Radius2 x 3.1416 x jV — cubic contents of A Laboratory Manual 
32 
X. 
Saliva. 
1. Add to some saliva in a test-tube a few 
drops of dilute (10%) acetic acid. Cloudi- 
ness due to mucin — not soluble in excess of 
the acid. 
2. Add to saliva a few drops HN03. A 
cloudiness which becomes yellow upon heat- 
ing. Cool and alkalinize with NaOII —orange 
color. This xanthoproteic reaction is due to 
mucin or an albuminoid bodv. 
3. To a small portion of saliva add an equal 
amount of water and a few drops Villon’s 
reagent. White pp., which becomes red upon 
warming. Due to mucin. 
4. Add NaOII and a few drops of a very 
dilute CuS04 solution. Violet color due to 
mucin. 
5. Add to a portion one drop of HC1 
and a few drops of a very dilute solution of 
ferric chloride, and shake thoroughly. Red 
color from the formation of ferric sulphocyan- 
ide. Saliva contains potassium sulphocyanide 
(KCNS), its presence being due to the gland 
containing adenin (C5HSN5), which is a poly- 
mere of hydrocyanic acid (CHN). Of Physiological Chemistry 
33 
6. Make a starch clyster (1 gramme starch to 
100 c.c. water), and, after cooling, take 10 c.c. 
of this solution, add 1 c.c. of saliva, and digest 
at 40° C. for an hour. Then put into a fermen- 
tation tube and allow to stand until the next 
day. It will be found that the resulting sub- 
stance (maltose) is fermentable. 
7. Perform the same test as in (6), but add 
to the starch and saliva mixture 1 c.c. digestive 
IICl. The action of the ptyalin is annulled. 
Prove by Trommer’s test and the fermentation 
tube. 
8. Determine the reaction of saliva. It is 
normally alkaline, but in pathological condi- 
tions, as in fevers or diabetes, it is acid. 
9. Examine some saliva under a micro- 
scope, and it will be found to contain certain 
morphological elements, such as salivary cor- 
puscles, pavement epithelial cells, and various 
fungi and bacteria. 
XI. 
Chemistry of Gastric Juice. 
Hydrochloric Acid. — 1. (a.) Add to some 
digestive hydrochloric acid in a test-tube a few 
drops of methyl-violet solution. Steel-blue 
color. 34 
A Laboratory Manual 
(b.) Make the same test with water. Violet 
color. 
(c.) Make the same test with lactic acid. 
Violet color, with slight shade of blue. 
2. Make the same test with HC1 solution, 
diluted 1 to 5. 
3. Take a portion of the HC1 solution in 
2 test-tubes. To one add an equal amount of 
water, to the other an equal amount of albu- 
mose-peptone solution ; then add methyl-vio- 
let to both and compare the color. 
4. Make the same tests with the IIC1 
solution diluted 1 to 5. The presence of large 
amounts of peptone makes methyl-violet use- 
less as a reagent. 
5. Add to :i portion of the lactic acid solution 
a few drops of methyl-violet, and divide into 
three parts. To No. 1 add an equal amount of 
water; to No. 2 add an equal amount 3% salt 
solution ; to No. 3 add an equal amount cone, 
salt solution. Nos. 1 and 2 change color but 
slightly. No. 3, however, becomes a distinct 
steel-blue. Sodium chloride disturbs the reac- 
tion in the presence of lactic acid only when 
strongly concentrated, due to the separation 
of the HC1 by the lactic acid. Of Physiological Chemistry 
35 
6. Evaporate a few drops of the mixtures 
formed under 2, 3, 4, and 5, in a small evapo- 
rating dish, to dryness over a free flame, 
avoiding too strong a heat. Intense blue resi- 
due. Peptones disturb the reaction only when 
present in large quantities. 
7. To a few drops of the digestive HCl in 
an evaporating dish add a few drops of tro- 
paeolin 00 solution, and warm gently. Lilac- 
blue color. 
8. Add to a few drops of digestive HCl 
in an evaporating dish a drop of Giinzburg’s 
reagent, and evaporate to dryness over a free 
flame, avoiding too strong a heat. Beautiful 
purple-red residue. Make the same tests with 
2, 3, 4, and 5. Peptone does not disturb the 
reaction and lactic acid does not give it. 
9. To a little IIC1 in a test-tube add a 
drop of dimethyl-amido-azo-benzol. Brilliant 
red color. 
10. Take two test-tubes, and in No. 1 place 
a few c.c. digestive HCl diluted 10 times ; in 
the other place an equal amount of water. To 
each add a drop of dimethyl-amido-azo-benzol. 
No. 1 becomes a brilliant red; No. 2 becomes 
cloudy and yellow. 
Lactic Acid. — The reagent used for the de- 
tection of lactic acid is Uffelmann’s, made by 36 
A Laboratory Manual 
taking 10 c.c. of a 5% carbolic acid solution and 
adding a few drops ferric chloride. Amethyst- 
blue fluid. 
1. To a portion of the reagent add a few 
drops digestive HC1. Decolorization. 
2. To another portion add some lactic acid. 
Canary-yellow color. 
3. To another portion add a mixture of 
equal parts of digestive HC1 and lactic acid 
solution. Canary-yellow color. With Uffel- 
rnann’s reagent lactic acid can be detected 
beside HC1, but not HC1 beside lactic acid. 
4. To Uffelmann’s reagent add some lactic 
acid and place in 3 test-tubes. To No. 1 add 
an equal amount of water; to No. 2 an equal 
amount cone. NaCl solution; to No. 3 an 
equal amount 3% NaCl solution. Only No. 
2 becomes decolorized. The presence of NaCl 
in small quantities does not disturb the de- 
tection of lactic acid. 
Pepsin. —1. Place in 3 test-tubes thin slices 
of hard-boiled egg of equal size. To No. 1 
add 10 c.c. digestive HC1; to No. 2 add 5 c.c. 
digestive 1IC1 with pepsin ; to No. 3 add 10 c.c. 
digestive 1IC1 with pepsin. Place all three 
in the brood oven for an hour or more, and 
note the difference in the thinning of the slices, 
due to the digestive action of the three fluids. Of Physiological Chemistry 
37 
2. Take the whites of 4 hard-boiled eggs, 
cut fine, and place in a fiask, adding 500 c.c. 
of the digestive HC1 with pepsin (1 gramme 
pepsin, 10 c.c. IICl, and water to 500 c.c.). 
Place in a brood oven for 2 to 3 days, shaking 
occasionally, filter, neutralize the filtrate, while 
warming gently, with Xa_,C03, and filter again 
from the acid albumen thus precipitated. Then 
evaporate to about 200 c.c., filtering again 
before reaching this point if more albumen 
becomes separated. The reaction meanwhile 
must be kept neutral by adding IICl or 
Na2C03, as the case should require. Now acid- 
ify the solution with acetic acid, saturate with 
NaCl, and filter. The filtrate contains pep- 
tones. Test for the same. 
XII. 
Clinical Analysis of tlie 
Stomach Contents. 
For clinical examination the stomach con- 
tents should be filtered, or if too thick and 
difficult of filtration, they should be diluted 
with a definite amount of distilled water. In 
quantitative estimations the amount should be 
measured with a pipette and not a graduate. 38 
A Laboratory Manual 
i. Presence of Pepsin. — Take 10 c.c. of 
the stomach contents, place in a test-tube, and 
add a thin slice of hard-boiled egg or fibrin. 
Place in a brood oven, and allow to remain a 
few hours. If after 10 hours there is no dim- 
inution of the size of the egg, or if there is 
an odor of decomposition, then pepsin is 
absent. If the stomach contents are weakly 
acid or alkaline, add an equal amount of diges- 
tive HC1. 
2. Presence of Rennet.— Take 5 c.c. neu- 
tralized stomach contents, and add to it an 
equal amount of cooked, neutral cow’s-milk, 
which has been fully cooled after cooking. 
Place in a brood oven, and if rennet be 
present, after 20 or 30 minutes the casein of 
the milk will have separated out in flocculent 
masses. 
3. Presence of Free HC1. — Use methyl- 
violet— tropaeolin 00—Giinzburg’s reagent or 
dimethyl-amido-azo-benzol. 
4. Quantitative Estimation of Total Acitl- 
ity.— Take 10 c.e. of the filtered stomach 
contents, add a few drops of rosolic acid until 
yellow, and titrate with a normal solution 
NaOH until a permanent rose-red color is 
obtained. 
C a 1 c u 1 a t i o n . — 1 c.e. NaOH — .00365 
grammes HC1, Of Physiological Chemistry 
39 
5. Quantitative Estimation of Total HC1 
(free and combined). 
Take 15 c.c. of the filtered stomach contents, 
add 1 gramme calcium carbonate, stir well, and 
filter. Take 10 c.c. of the filtrate, place in a 
small flask, and pass air through until the 
C02 is all driven off. Then add 5 c.c. cal- 
cium chloride, a few drops of rosolic acid, and 
titrate with normal NaOII as before. The 
difference between the acidity found before and 
that found now equals the total HC1 (free and 
combined). 
1 c.c. NaOH = .00365 grammes HC1. 
The principle of this method is based upon 
the fact that the calcium carbonate combines 
with the free and combined HC1 and the 
organic acids to form neutral calcium salts, 
while the acid phosphates are not affected; 
and by determining as in (4) the total acidity, 
and deducting from this the acidity due to the 
acid salts, the amount of total ITC1 is obtained. 
G. Test for lactic acid by Uffelmann’s re- 
agent. 
7. Acetic, Butyric, and Formic Acids. — Dis- 
til a portion of the stomach contents, and, if 
present, these acids will be found in the dis- 
tillate. 
(«.) Test for Acetic Acid. —Accurately neu- 4° 
A Laboratory Manual 
tralize a portion of the distillate with NaOH 
and then add ferric chloride. Red color, if 
present. 
(5.) Tests for Butyric Acid. — 1. Add ferric 
chloride. Red color, which disappears on heat- 
ing. 2. Add cone. H2S04, and warm. Charac- 
teristic odor of rancid butter. 
(c.) Test for Formic Acid. — Add silver nitrate. 
White pp., which turns black in the dark. 
8. Albumose Peptone. — Neutralize, while 
warming, with Na2C03, filter, and test filtrate 
by biuret test. 
9. Starch and Erythrodextrine. — Test with 
a solution of iodine in potassium iodide. They 
should not be present an hour after food is 
taken. 
lO. Resorptive Power of the Stomach. — 
Give a capsule containing 0.1 gramme potas- 
sium iodide, and test the saliva every few 
minutes with filter-paper moistened with starch 
solution until a blue color is obtained. It 
ought to be absorbed in from 8 to 15 minutes in 
a normal stomach. 
11. Motor Power of the Stomach. — A 
capsule containing 1 gramme of salol is given, 
and separate portions of the urine, passed within 
intervals of half an hour, are tested by ferric 
chloride. The violet color, due to salicyluric Of Physiological Chemistry 
41 
acid, is obtained, under normal conditions, in 
from 45 to 75 minutes. 
Mieroseopic Examination. 
Sarcinae. — They occur in the form of pecul- 
iar colonies of cocci, arranged in squares, and 
strongly resembling cotton-bales. In normal 
conditions they are found only in small num- 
bers. 
Yeast-Fungi — have a characteristic oval 
form, arranged in buds. They are diagnostic 
of fermentative dyspepsia. 
XIII. 
The Bile. 
Biliary Acids (Glycocholic and Taurocholic), 
Pettenkofer’s Test. — Take a few c.c. of bile, 
add an equal amount of water and a few drops 
of cane-sugar solution. Then pour one-half 
the amount of cone. II2S04 down the side 
of the test-tube, so that the latter shall under- 
lie the bile. At the juncture of the bile and 
the acid there is found a purple-red zone. 
Then dip the test-tube into a glassful of water, 
and mix the two fluids carefully by shaking or 
swaying the tube to and fro (if the resulting 42 
A Laboratory Manual 
heat is developed too suddenly the test is 
spoiled), and we obtain a magnificent purple- 
red fluid. 
After the tube is fully cooled, pour one-half 
into another tube containing alcohol, and the 
second half into a third tube containing glacial 
acetic acid. 
(a.) The acetic acid solution shows in the 
spectroscope a line in green. 
(b.) The alcoholic solution shows at first 
only the line in green, but the solution soon 
becomes brownish, and we find a line in blue. 
The results of these tests show that we have 
present both glycocholic and taurocholic acids, 
as well as cholic acid, which forms the basis of 
both. 
This test can be modified by diluting the 
bile tenfold, and taking a few drops of the 
dilute solution, and adding a trace of sugar and 
a few drops cone. H2S04. Evaporate in an 
evaporating dish upon the water-bath, and a 
violet-red color appears upon the dish at the 
edge of the fluid. As soon as this appears 
cease evaporating. 
Biliary Pigments (Bilirubin and Biliver- 
din), Gmelin’s Test.—Dilute some of the bile 
ten times, take a portion in a test-tube, and 
pour down the side of the tube some impure Of Physiological Chemistry 
43 
HN03 (nitric acid containing HN02), so 
that the acid shall underlie the bile. There is 
formed at the junction of the two fluids a play 
of colors, consisting of green, blue, violet, red, 
and reddish-yellow. The green must always 
be present. The play of colors in this test is 
the result of oxidation, the part nearest the 
acid being most intensely oxidized. 
Huppert’s Test. — Take some dilute bile 
solution, and add lime-water as long as a pp. 
is formed. Filter the pp. oft* (which consists 
of bilirubin lime), wash once with water, and 
place while still moist in a test-tube filled half 
full with alcohol which has been acidified with 
sulphuric acid. Heat to boiling in the water- 
bath. The fluid takes on a brilliant green or 
bluish-green color. 
XIV. 
Pancreatic Juice. 
Trypsin. — Dissolve 1 gramme powdered 
extract of pancreas in 500 c.c. water, and add 
5 c.c. of a saturated sodium carbonate solution. 
Place in a flask, and add the whites of two hard- 
boiled eggs finely chopped. Add a few drops 44 
A Laboratory Manual 
of chloroform, cork the flask tightly, and place 
in a brood oven for a few days, shaking occa- 
sionally. Now acidify carefully with acetic 
acid while warming, and filter. Shake a portion 
of the filtrate in a test-tube with bromine 
water. It should give a violet-red color. Now 
evaporate the filtrate to about 100 c.c. and allow 
to stand in a cool place until the next day. 
Separation of white crystals of tyrosin. Filter 
these off (preserving the filtrate) and examine 
under the microscope. Colorless, needle-shaped 
crystals of tyrosin. Dissolve some in water in 
test-tube, add a fewT drops of Millon’s reagent, 
and heat to boiling. Rose-red to deep-red color. 
Scherer’s Test.—Evaporate a few of the 
tyrosin crystals to dryness on platinum foil 
wfith nitric acid. A yellow residue is obtained, 
which on adding NaOH develops a deep red- 
dish-yellow color. 
The filtrate from the above is to be evapo- 
rated still further upon the water-bath, until 
there is formed a scum upon the surface of the 
fluid. This scum is leucin. Examine under 
the microscope. Crystals of leucin, which 
consist of globules having a pearly lustre and 
radial lines. Easily soluble in IIC1 and 
alkalies. 
Diastase. — Add 1 gramme pancreas powder Of Physiological Chemistry 
45 
to 50 c.c. water, digest for two hours at 40° C., 
and filter. Take equal parts of the filtrate and 
starch clyster (made as for saliva) and digest 
for a short time in the brood oven. The starch 
solution becomes transparent and shows the 
presence of sugar by Trommer’s test. PART II. 
THE URINE. THE URINE. 
L 
General Characteristics. 
Pale. 
Normal. 
High. 
c5 
Color. — Normal 
Blood. 
Bile. 
Chyle. 
Pathological 
Accidental — due to drugs. 
© 
Odor. — Accidental (due to drugs). Uri- 
nous. Ammoniacal. Due to acetone. 
Reaction. — Acid or alkaline. The alkali 
may be fixed or volatile. If fixed, it does not 
disappear on heating ; if volatile, it disappears 
on heating. 
Specific Gravity. — Normal, 1015—1025 
Amount. — Subject to great variation. Nor- 
mal, 1,500 c.c. 
Solids by Specific Gravity. — Multiply last 
two figures of specific gravity by 2.33, which 
gives the number of grammes of solids in 1,000 
e.c. 50 
A Laboratory Manual 
IL 
General Chemistry and 
Clinical Analysis. 
Urobilin. — Take a little H2S04 in a test- 
tube and pour on it, from a height, some urine. 
Purplish-brown color, if present. 
If decreased — light color. 
If normal — dark and transparent. 
If increased — dark and opaque. 
Indican (Stokvis’ Test). — To 1 test-tube of 
urine add an equal amount HC1 and one or 
two drops calcium hypochlorite, and shake. 
Then add a few drops of chloroform and shake 
thoroughly. If indican is present, the chloro- 
form will be colored blue. If normal, it will 
be about the color of litmus. 
Albumen (Qualitative). 
Serum Albumen. Nitric Acid Test. — To 
about | wine-glassful of urine add with a 
pipette about t the amount of cone. IIX03, 
so that the acid shall underlie the urine. If 
present, there will be a distinct white zone at 
the point of contact, which is sharply defined 
and extends either way. If bile be present, Of Physiological Chemistry 
51 
the albumen zone will be tiimed oreen. In 
© O 
the nitric-acid test several other zones will be 
present, viz. : 
1. A nitrate of urea zone, which -always 
occurs in the nitric acid and extends down- 
wards. 
2. A zone of indican and urobilin, which is 
exactly below the albumen zone. 
3. A zone due to urates, which appears above 
the nitric acid in the urine and extends up- 
wards. 
4. A mucin zone in the upper part of the 
urine. 
Heat Test. — To some filtered urine in a test- 
tube add a drop of dilute acetic acid, and heat 
the upper part of the fluid. If present, there 
will be a cloudiness to a coagulation, which can 
be best seen by comparing it with the clear 
fluid below. This test, if properly performed, 
is a very delicate one. 
aSTucleo-Albumen, — Add a few drops of 10% 
acetic acid. If present, there will be a pp. 
soluble in excess of the acid. 
Mucin. — Same as for nucleo-albumen, but not 
soluble in excess of acetic acid. 
Globulin. — Dilute a small amount of urine 
with several times its bulk of water. If pres- 
ent, a pp. will be formed. 52 
A Laboratory Manual 
Albumose-peptone. — Remove the other albu- 
mens by 10 % acetic acid and heating; filter, 
and test filtrate by : (a) Biuret reaction. (b) 
Take 10 c.c. of above urine, add 1 c.c. HC1, and 
then precipitate with phospho-tungstic acid. 
Warm slightly, let pp. fall to the bottom of the 
tube, pour off the fluid above, and wash pp. 
with water. Dissolve pp. in NaOH and add 
a weak solution CuS04. Biuret reaction. 
Albumen (Quantitative). 
Esbacli’s Test. — Fill to the mark U with 
clear, filtered urine, and then add Esbach’s re- 
agent to the mark R. Let stand for 24 hours 
or shake down in a centrifuge. Every mark 
occupied by the pp. represents A- of 1 % of 
albumen, or 1 gramme to the liter. 
S u gar (Qua! it at i ve ). 
Trommer’s Test. — Take test-tube of urine, 
add an equal amount of NaOH, and then add 
CuS()4 until it no longer dissolves. Heat the 
upper part, and if sugar be present there will 
be a reduction of the copper to the red 
suboxide. 
Feliling-’s Test. —Take a few c.c. of Fehling’s 
solution, dilute with about 4 times the amount 
of water, and heat to boilino;. Then add a Of Physiological Chemistry 
53 
little urine and heat once more near the upper 
part of the fluid. Reduction of copper, if sugar 
is present, to red suboxide. 
Nylander’s Test. — Add to the urine about 
the amount of Nylander’s solution, and boil. 
Reduction of metallic bismuth if sugar is pres- 
ent. 
S ugar ( Quantitative). 
Fehling’s Method. —First ascertain the spe- 
cific gravity. 
If 1.030 or below, dilute 5 times, by taking 
10 c.c. of urine and adding 40 c.c. water. 
If above 1.030, dilute 10 times, by taking 5 
c.c. urine and adding 45 c.c. water. 
Place the urine in a burette. Take 10 c.c. 
Fehling’s solution, place in a small flask, and 
add 40 c.c. water. Bring to a boiling point, 
and add the urine a little at a time, boiling 
between each addition. Continue this until 
the blue color has entirely disappeared. 
Calculation.—10 c.c. Fehling’s solution = 
.050 grammes sugar; therefore the amount of 
mixed urine used in the titration, divided by 
its degree of dilution, contains exactly .050 
grammes sugar. Calculate entire amount for 
24 hours. 
Fermentation Test. — Ascertain accurately 
the specific gravity of the urine. Then take 50 54 
A Laboratory Manual 
c.c. of the urine and add 2 grammes yeast. 
Place in a bottle which is closed tightly and 
through the cork of which is passed a reversed 
pipette. Place in a warm place and allow to 
stand for 24 hours. Again take the specific 
gravity after fully cooled, and the difference 
multiplied by 230 gives the percentage of 
sugar. 
Note. — In all quantitative estimations albu- 
men must be removed, and the urine measured 
with a pipette and not a graduate. 
Urea (Qualitative) 
To a few drops of urine on a microscopic 
slide add a drop of cone. HN03, and in a 
few minutes crystals of urea nitrate will have 
separated out. Examine with a microscope. 
Quantitative (Liebig’s Method). 
Take 20 c.c. urine and add to it 10 c.c. 
baryta mixture. Then filter, take 15 c.c. of 
the filtrate, which should be clear, place in a 
beaker, and titrate with a standard solution of 
mercuric nitrate, until a drop, added to a 
saturated solution sodium carbonate, gives a 
yellow pp. of oxide of mercury. 
Calculation.— 1 c.c. mercuric nitrate = .010 
grammes urea. Gives amount of urea in 10 Of Physiological Chemistry 
55 
c.c. urine. Calculate entire amount for 24 
hours. 
Normal amount, 25 to 40 grammes in 24 
hours. 
Uric Acid (Qualitative) 
Take 20 c.c. urine and add to it 2 c.c. cone. 
HC1. Set aside for 24 hours in a cool place. 
The uric acid which has separated out is col- 
lected on a filter and subjected to the murexid 
test. 
Murexid Test. — Place the crystals in an 
evaporating dish, and dissolve them in a few 
drops of HN03 with the application of heat. 
Evaporate carefully to dryness, when a yellow- 
ish-red spot will be found to remain. Cool, and 
add a drop of ammonia, when a beautiful pur- 
plish-red color will develop, owing to the 
formation of ammonium purpurate (murexid). 
Quantitative (Hopkins’ Method). 
Take 100 c.c. urine and add 30 grammes of 
finely powdered ammonium chloride. The 
mixture is then allowed to stand for two hours, 
and is stirred from time to time. Then filter 
and wash the pp. two or three times with a satu- 
rated solution (NH4)C1. Wash the pp. from 
the filter, with a little boiling water, into an 56 
A Laboratory Manual 
evaporating dish, and add 5 c.c. HC1. Evapo- 
rate the solution until crystals of uric acid 
begin to separate out. These are collected on 
a filter, the filter-paper with the adherent pp. 
is placed in a beaker, and the crystals are 
dissolved in as small an amount of a 30 % solu- 
tion of sodium carbonate as possible, with the 
aid of heat. Cool, dilute with water to 100 
c.c., add 20 c.c. cone. H2S04, and warm. 
Titrate, while warm, with a 0¥ normal solution 
potassium permanganate, until the rose-red 
color no longer disappears on stirring. 
Calculation. — 1 c.c. potassium permanga- 
nate ==3.61 milligrammes uric acid. Gives 
amount of uric acid in 100 c.c. urine. Calcu- 
late entire amount for 24 hours. Normal amount 
eliminated in 24 hours, about 1 gramme. 
Chlorides (Qualitative). 
To a few c.c. urine which has been acidified 
with a few drops HN03 add a few drops AgN03. 
White pp. of silver chloride. 
Quantitative (Mohr’s Method) 
Take 10 c.c. urine, acidify with a few drops 
HXOg, and dilute with distilled water to 50 
c.c. Then add very carefully sodium earbo- Of Physiological Chemistry 
57 
nate in substance until just neutral. Then 
add a few drops yellow K2Cr04, which gives 
the solution a yellow color, and titrate with a 
standard solution of silver nitrate until a distinct 
orange tinge is obtained after brisk stirring. 
Calculation. — 1 c.c. AgN03 = .010 gramme 
chlorides. Gives amount of chlorides in 10 
c.c. urine. Calculate entire amount for 24 
hours. 
Normal amount eliminated in 24 hours, 
11-15 grammes. 
Sulphates. 
To a few c.c. of urine add a few drops of 
solution of barium chloride and HC1. A white 
pp. of barium sulphate. 
Total Phosphates. — A few c.c. of urine are 
acidified with a few drops of acetic acid and 
then treated with a solution of ferric chloride. 
A yellowish-white pp. of ferric phosphate. 
Phosphates (Qualitative) 
Earthy Phosphates. — To a few c.c. of 
urine add ammonia in excess. A flocculent 
pp. indicates their presence. 
Alkaline Phosphates.— Precipitate the earthy 
phosphates with NH4OH, filter, and to the clear 
filtrate add magnesium sulphate solution, A 
heavy white pp. 58 
A Laboratory Manual 
Quantitative Estimation of Phos- 
phoric Acid. 
Take 50 c.c. urine and add 5 c.c. of sodium 
acetate solution. Heat the mixture to boiling, 
and titrate with a standard solution of uranyl 
nitrate, until a drop, placed in a solution 
of potassium ferrocyanide, gives rise to a 
mahogany-brown pp. 
Calculation. — 1 c.c. uranyl nitrate — .005 
gramme P2Os. Gives amount of phosphates 
in 50 c.c. urine. Calculate entire amount for 
24 hours. 
Normal amount eliminated in 24 hours, 
1-2 grammes. 
Bile Pigments. 
Gmelin’s or Huppert’s Test.— Green must 
always be present. 
Determine with a spectroscope which color- 
ing matter is present. 
Guaiac Test. — To a few c.c. of urine add a 
few drops of freshly prepared tincture guaiac 
and a little old oil of turpentine. Shake thor- 
oughly, and if blood is present the mixture 
becomes blue, 
Blood Coloring Matters. Of Physiological Chemistry 
59 
Kreatinin. 
A few c.c. of urine are treated drop by drop 
with a very dilute solution of sodium nitro- 
prusside, and then drop by drop with a solu- 
tion NaOII. In the presence of kreatinin a 
ruby-red color develops, which is best seen in 
the lower portion of the tube. Then add a few 
drops glacial acetic acid and warm, and the 
color changes to green. 
© © 
Acetone. 
1. Same test as for kreatinin, except that 
on the addition of acetic acid a purple instead 
of a green color develops. This takes place 
without warming. 
2. A few c.c. of urine are treated with a 
few drops of iodine in potassium iodide solution 
and NaOH. Warm, and a pp. of iodoform 
occurs, which may be recognized by its odor. 
If these reactions for acetone fail, take about 
1 liter of urine, add a few drops H2S04 and 
distil about £, and then redistil about 10 c.c. 
As acetone is very volatile, if any is present 
it will be found in the 10 c.c. 
Aceto-Acetic Acid. 
Add ferric chloride solution. There is a pp. 
of ferric phosphate formed, which is allowed to 6o 
A Laboratory Manual 
settle. On the further addition of ferric chlo- 
ride, a purple color is formed if the reaction 
is positive. 
ElirliclTs Diazo-Reaction 
To a few c.c. of urine add an equal amount 
of sulphanilic acid and a few drops of sodium 
nitrite. Shake thoroughly, and then allow 1 
or 2 c.c. of ammonia to run down the side of 
the tube so as to overlie the fluid below. If 
the reaction is positive, a carmine-red ring 
develops at the junction of the two fluids. 
This reaction takes place in febrile urines, and 
is due to a peculiar chromogen. The test wTas 
former!}7 thought to be especially diagnostic of 
typhoid fever. 
m. 
Urinary Sediment.1 
Unorganized Sediments. 
Uric Acid.—Whetstone shape. Almost 
always colored yellow or brown. Forms the 
brick-dust sediment. Soluble in NaOH. 
1 To obtain a urinary sediment, allow the urine to stand in a 
conical glass over night, or, what is better, throw it down with the 
centrifuge, and with a pipette transfer a small portion to a slide, 
cover with a cover-glass, and examine with a high power. Do not 
use a condenser. Of Physiological Chemistry 
61 
Urates. —Sodium and potassium are amor- 
phous. Ammonium, dumb-bell shape, or round, 
or sheaf-shaped. 
Oxalate of Calcium. — Envelope, dumb-bell 
or oval shape. Insoluble in acetic acid. 
Phosphates. —Coffin-lid shape. Soluble in 
acetic acid. 
Cystin is very rare. Small, colorless, hex- 
agonal crystals, soluble in ammonia. 
Hippuric acid (in long needles), leucin 
(spherules with concentric striations), and ty- 
rosin (in fine needles) are not common. 
Organized Sediments. 
Blood.— Normal and abnormal shapes of 
corpuscles; that is, round or shrivelled 
(crenated). 
Leucocytes. — Resemble distended red blood 
corpuscles. 
Pus Corpuscles.— Are round, with nuclei 
and granules. On the addition of acetic acid 
the granular matter is destroyed and the nuclei 
are brought out very clearly. 
Epithelial Cells.—Are either normal, gran- 
ular, or fatty. These three conditions mean 
simply the age of the process. 
Renal (from convoluted tubules) — are 
slightly polygonal, with nucleus or with gran- 
ules, nucleus persisting. 62 
A Laboratory Manual 
Renal (from straight tubules) — are less 
polygonal and more circular. 
Pelvic — battledore shape, if superficial; 
round, if from the deeper layers. 
Ureter — spindle-shaped cells. 
Bladder — from fundus polygonal; from 
neck round. 
Vaginal — large and very faint, frequently 
overlapping. 
Urethral — spindle-shaped. 
Prostate — resemble pelvic cells (deep). 
Fat may be found as free fat, or dotting 
epithelial cells, or as so-called compound 
granular cells. 
Casts. 
Hyaline — faint, with both ends rounded. 
Fibrinous — contains a substance of greater 
density. Varies from hyaline only in intensity. 
Blood — cast covered with blood corpuscles 
and leucocytes. 
Epithelial — cast covered with renal epithelial 
cells. 
Granular — cast covered with granules. 
Fatty — cast covered with fat globules. 
Waxy—are very distinct, intense, and homo- 
geneous. Slightly yellow. Of Physiological Chemistry 
63 
Fungi and Bacteria. 
Micrococcus Ureae. Produces ammoniacal 
fermentation, converting urea into ammonia. 
Bacterim Termo. Rod-like shape. 
Pencillium Glaucum. Grows in chains. In- 
dividuals are oval-shaped, with a little depres- 
sion at each end. Occurs only in acid urines. 
Yeast Fungi. Found only in diabetic urines, 
or in urines containing sugar. Ovals with de- 
pressed centres. 
Sarcinae. Analogous to the sarcinae found 
in the stomach. 
Tubercle Bacilli. Found in tuberculosis of 
the genito-urinary tract. Do not confound 
with the smegma bacillus, which morphologi- 
cally resembles the tubercle bacillus. 
Spermatozoa. PART III. 
TOXICOLOGY. I. 
Acids. 
If unmixed with organic matter, concentrate 
if too dilute. 
Sulphuric Acid. 
A. First determine whether a free acid 
or a sulphate is present, by methyl-violet or 
dimethyl-amido-azo-benzol. 
1. To a small portion of the fluid add 
acetate of lead solution. White pp. 
2. To another portion add barium chloride 
solution. Heavy white pp. 
3. To another portion add calcium chloride 
solution. Insoluble white pp. 
B. If mixed with organic matter (stomach 
contents, etc.), concentrate to a syrup, extract 
with successive portions of alcohol, and drive 
off the alcohol by warming on the water bath. 
A watery solution is left, and with it perform 
tests 1, 2, 3. 
Nitric Acid. 
A. If unmixed, test for free acid, to dis- 
tinguish from nitrates. 68 
A Laboratory Manual 
1. Boil a small portion with a little albumen 
(white woolen thread or peptone) — yellow 
color. Cool, and add NaOH — orange color. 
( Xanthoproteic reaction.) 
2. Add to a small portion in a test-tube a 
piece of copper and some concentrated sulphuric 
acid, and heat. Red fumes. 
3. Add to a portion some sulphuric acid, and 
pour carefully down the side of the tube some 
ferrous sulphate solution. Brown zone at point 
of contact. 
B. If mixed with organic matter, concen- 
trate, cool, extract rapidly with alcohol, neu- 
tralize with potash, tilter, and drive off the 
alcohol. Use tests 2 and 3. Reacts equally 
well with nitrates. 
Hydrochloric Acid. 
A. Tf unmixed, test for free acid. 
1. Add silver nitrate. AVhite pp. soluble 
in ammonia, which darkens on exposure to 
light, 
2. Add lead acetate solution. Heavy, 
white, crystalline pp. 
3. Add mercurous nitrate solution. Fine 
white pp. 
B. Tf mixed with organic matter, distil, 
and test distillate. Of Physiological Chemistry 
69 
Oxalic Acid. 
Does not react to tests for free mineral acids. 
A. If unmixed : 
1. Add calcium chloride or calcium sulphate 
solution. Fine powdery pp. of calcium oxa- 
late. Examine with the microscope. 
2. Add silver nitrate solution. White pp. 
of silver oxalate, soluble in ammonia, and with 
some difficulty in nitric acid. Silver chloride 
is insoluble in nitric acid. 
3. Add baryta water. White pp. 
B. If mixed with organic matter, acidulate 
with IIC1; evaporate to dryness, extract hot 
(b}r boiling) with alcohol; drive off the alcohol 
and dissolve residue in water. Apply all the 
tests. 
Carbolic Acid. 
Does not react to tests for free mineral acids. 
A. If unmixed: 
1. Add ferric chloride solution. Deep 
amethyst-blue color. 
2. Add to a small portion i its volume of 
ammonia, a few drops calcium hypochlorite 
solution, and warm gently, but not to boiling. 
Blue to green color. 
3. Add a few drops Millon’s reagent and 
heat to boiling. Intense dark-red color. 70 
A Laboratory Manual 
4. Add bromine water. A gelatinous pp. of 
tribromophenol, soluble in excess of carbolic 
acid. 
B. If mixed with organic matter, acidify 
strongly with HC1 and distil. Apply al 1 the tests. 
Hydrocyanic Acid (Potassium Cyanide) 
A. If unmixed with organic matter : 
o 
1. Add a few drops ferrous sulphate solution, 
then a few drops of ferric chloride, and acidify 
strongly with HCl. Blue pp. of Berlin blue. 
2. Add some ammonium sulphide, evaporate 
to dryness upon the water bath, acidify with 
HCl, and add a couple drops of ferric chloride. 
Blood-red color from sulphocyanide of iron. 
3. Add a few drops of picric acid, and warm. 
Blood-red color from isopurpurate of potash. 
4. Add silver nitrate. White pp. of silver 
cyanide. 
B. If mixed with organic matter : 
1. Place a piece of filter-paper moistened 
with guaiae mixture in the cork of the flask, 
and warm. Filter-paper becomes blue. 
2. Acidify with tartaric acid and distil into 
a weak potash solution. The delivery tube 
must pass below the surface of the solution, to 
prevent escape into the air. With the dis- 
tillate perform the tests. Of Physiological Chemistry 
71 
IL 
Metallic Poisons. 
Arsenic (Arsenious Acid). 
1. Place a small granule of white arsenic in 
the closed end of a reduction tube, and above 
this place a small splinter of charcoal. First 
heat where the charcoal is, then the closed end 
of the tube, and just above the charcoal there 
is formed a mirror of metallic arsenic. Now 
remove the charcoal and heat the mirror, and a 
sublimate of white arsenic is formed above it. 
Examine with a microscope. Octahedral crys- 
tals. 
A. If unmixed with organic matter : 
2. Slightly acidify some of the solution with 
HC1, and pass II2S through it. Yellow sul- 
phide of arsenic is formed, which is soluble in 
ammonia. 
3. Add ammonia to some of the solution, 
drive off the excess by warming on the water 
bath, and divide into 2 parts. To : 
(a.) Add silver nitrate, and a yellow pp. 
of silver arsenate is formed. To : 
(b.) Add copper sulphate. A pale green 
pp. of arsenate of copper (Sclieel’s green). 72 
A Laboratory Manual 
B. If mixed with organic matter : 
Reinscli Test. — Strongly acidulate the solu- 
tion with HC1, place in it a piece of bright 
copper tinsel, and warm 10 to 15 minutes over 
the water bath. The copper, which has become 
covered with a grayish-white layer, is now to 
be removed, washed with water, alcohol, and 
ether, and thoroughly dried. Place in a small 
reduction tube and heat gently. Sublimate of 
arsenious acid formed in the cool part of the 
tube. Examine with the microscope. Octa- 
hedral crystals. This method, although not as 
delicate as the Marsh test, possesses the advan- 
tage that it can be applied in the presence of 
organic matter. 
Antimony (Tartar Emetic) 
A. If unmixed : 
1. Add to some of the solution its vol- 
ume of IIC1 and place in the solution a piece 
of tin. Black deposit of antimony upon the tin 
in the cold. 
2. Acidulate some of the solution with IIC1 
and pass II S through it. Orange-red pp. of 
sulphide of antimony. 
3. Add nitric acid drop by drop to some of 
the solution. White pp. of subnitrate of 
antimony, soluble in excess of nitric acid. Of Physiological Chemistry 
73 
Ik If mixed with organic matter : 
Fresenius-Babo Method. — Acidify strongly 
with IIC1 (| its volume), and add, while warm- 
ing, chlorate of potash, until the solution is 
yellow. Drive off the free chlorine, filter, 
dilute largely with water, and pass II2S through. 
Orange-red pp. of sulphide of antimony. As 
arsenic may give a somewhat similar pp. and 
organic matter still be present, filter out the 
pp. dry, and burn with saltpeter mixture. After 
cooling, dissolve the residue, with warming, in 
dilute IIO (1: 2), and to the clear solution 
apply the individual tests. 
Mercury (Corrosive Sublimate). 
A. If unmixed : 
1. Pass H2S through a small portion, and 
there is formed a pp. which is at first yellow 
and finally black. 
2. Add XaOH to some of the solution. 
Yellow pp. of mercuric oxide. 
3. Add (XH4)OH. White pp. of aniido- 
mercuric chloride, soluble in HC1. 
4. Add subchloride of tin, and there is 
formed a white pp. of subchloride of mercury 
or a gray one of mercury. 
5. Add potassium iodide. Scarlet pp. of 
mercuric, iodide. 74 
A Laboratory Manual 
B. If mixed with organic matter: 
Acidify with IK I, insert a piece of bright 
copper tinsel, and warm over the water-bath. 
Remove the copper, wash, dry, and heat in 
small ignition tube. Sublimate of mercury 
formed in the cool part of the tube. Place a 
small granule of iodine above the sublimate in 
the tube, and heat the sublimate once more. 
Bright red zone of iodide of mercury. Ex- 
amine with the microscope. Diamond-shaped 
crystals. 
m. 
Metallic Poisons (Continued) 
Copper (Copper Sulphate). 
A. In pure solution : 
1. Pass H2S tlirough the solution. Black 
sulphide of copper. 
2. Add some potassium ferrocy anide. 
Brownish-red 
Add ammonia to some of the solution. 
Blue pp., soluble in excess of the ammonia, 
giving a deep-blue fluid. 
B. If mixed with organic matter : 
1, Accidulate with HC1 and insert a piece Of Physiological Chemistry 
75 
of steel. Bright-red layer of copper upon the 
steel. 
2. Destroy organic matter by HC1 and 
KCIO3 (Fresenius-Babo), filter, dilute filtrate, 
and pass H2S through. Black pp., which can 
be further verified by the above tests. 
Lead (Lead Acetate). 
A. In pure solution : 
1. Add sulphuric acid. White pp. of lead 
sulphate. 
2. Add HC1. White pp. of lead chloride, 
insoluble in ammonia. (Distinction from 
silver chloride.) 
3. Add potassium ferrocyanide. White 
pp. of ferrocyanide of lead. 
4. Add potassium iodide. Yellow pp. of 
lead iodide. 
B. If mixed with organic matter : 
Destroy organic matter with HC1 and 
KC103, filler while hot, and, upon cooling and 
diluting, white crystalline lead chloride may be 
deposited. This can be redissolved in hot 
water and the tests applied. If the chloride 
is not deposited, pass H2S through the solution, 
filter off the black lead sulphide, and dissolve 
in warm nitric acid. Verify by the tests. 76 
A Laboratory Manual 
Phosphorus. 
1. Fasten a piece of filter-paper, moistened 
with silver nitrate solution, to the cork of the 
flask containing the mixture, and warm slightly 
(by placing the flask in warm water). Filter- 
paper becomes blackened, due to the formation 
of silver phosphide. This must take place in 
the dark, as silver nitrate is blackened by light 
alone. It is also best to place a second piece 
of filter-paper in the cork, moistened with lead 
acetate, to exclude hydrogen sulphide, which 
would also blacken silver nitrate. 
2. Acidify a portion with sulphuric acid, 
and distil into a weak solution of silver nitrate. 
If examined in the dark, the vapor is phos- 
phorescent in the cooler, and the silver is pre- 
cipitated as a black phosphide of silver. 
Nitro-Benzol. 
A. If unmixed : 
1. Mix some of the solution with alcohol, 
add NaOH and some sulphur or the sulphide of 
an alkali. Red color, if present. 
2. Distil a portion, dissolve the oily drops 
of the distillate in alcohol, and treat with zinc 
and dilute II2S04. From this aniline is formed. 
Render alkaline with XaOlI, shake with ether, 
allow the ether to evaporate, and treat the resi- Of Physiological Chemistry 
77 
due with chloride of lime solution. Violet 
color. 
3. Distil a portion, add to distillate a few 
drops KOH, boil, and then add carbolic acid. 
Evaporate to a ring of dryness, and touch the 
ring with calcium hypochlorite solution. Green 
color, if present. 
B. If mixed with organic matter, distil, 
and apply all the tests. 
For the detection and the isolation of the 
metallic poisons when mixed with organic 
matter, the ideal method is that of Fresenius- 
Babo ; that is, the destruction of organic matter 
with hydrochloric acid and potassium chlorate, 
as given above. In the metals under consid- 
eration it can be applied to antimony, copper, 
lead, arsensic, and mercury. As the two latter 
are volatile, care must be taken against their 
possible loss, by conducting the operation in 
a flask containing a cork with a long tube, 
instead of in an evaporating dish, as with anti- 
mony, copper, and lead. In the case of 
arsenic, the latter procedure is not absolutely 
necessary, provided care be taken to add the 
potassium chlorate before heating. 78 
A Laboratory Manual 
IV. 
Alkaloids. 
If unmixed with organic matter : 
Morphine. 
1. Take 1 or 2 c.c. of the solution in a 
test-tube, acidify with sulphuric acid, add a 
couple drops of chloroform, a small granule of 
iodic acid, and shake thoroughly. Chloroform 
becomes colored red, showing the presence of 
morphine. 
2. Take a granule of the substance (or if 
in pure solution, evaporate to dryness on the 
water-bath), add dilute nitric and sulphuric 
acids (a drop of each only), warm, and allow 
to cool. Blood-red color. 
3. Dissolve a granule in a couple drops of 
nitric acid. Orange-red color. 
4. Add to a granule a drop of Froehde’s 
reagent. Colored violet, green, blue-green, 
and yellow. 
Strychnine. 
1. Dissolve a granule or two in concen- 
trated sulphuric acid in a porcelain dish, and 
then add a small Granule of red chromate of Ot Physiological Chemistry 
79 
potash. ( olored blue, violet, and finally 
cherry-red. 
2. Add a drop of dilute nitric acid to a 
granule, in an evaporating dish, and warm 
carefully. Now add to the warm solution a 
small crystal of chlorate of potash. Intense 
scarlet color, which changes to brown upon 
the addition of 1 or 2 drops of ammonia, with 
the formation of a brown pp. 
Yeratrine. 
1. V arm with hydrochloric acid. Beauti- 
ful red color. 
2. Add to some of the dry alkaloid some 
cane-sugar, and moisten with concentrated sul- 
phuric acid. ellow, blue, and finally a violet 
color. 
3. "With kroehde’s reagent. Yellow, then 
cherry-red. 
Atropine 
1. Dissolve a portion of the solid in fum- 
ing nitric acid, evaporate to dryness upon the 
water bath, and moisten, after cooling, with a 
10% alcoholic KOH solution. Violet, then 
cherry-red color. 
2. Heat a portion of the dry alkaloid in a 
small closed tube, or in an evaporating dish, 8o 
A Laboratory Manual 
over a free flame, until white fumes begin to 
rise. Odor of violets. 
Cocaine. 
1. Add 5% chromic acid solution to a por- 
tion, and there is formed a pp. which at first 
becomes redissolved, but after a time returns 
as a crystalline orange-colored pp. of cocaine 
chromate. This reaction takes place more 
quickly upon the addition of a few drops of 
cone. HC1. 
2. Add 5 % chromic acid solution, and warm. 
Green color, with fumes of benzoic acid. 
3. Add drop by drop 1 % potassium per- 
manganate solution. Small violet crystals of 
permanganate of cocaine. 
4. Touch a small portion of a dilute solu- 
tion to the tongue, and there is caused a numb- 
ness lasting for some time. 
if the alkaloid is mixed with organic matter, 
in order to isolate it it is necessary to resort 
to the method of Stas-Otto. 
The Method of Stas-Otto 
1. Evaporate upon the water-bath to a 
syrup. 
2. Transfer to a flask containing a cork with Of Physiological Chemistry 
81 
a long tube, add twice as much of a 1 °/0 alco- 
holic tartaric acid solution, and warm upon the 
water-bath from 30 minutes to an hour. 
3. Cool, filter, concentrate the filtrate until 
the alcohol is driven oft‘, and filter once more 
through a filter moistened with water. 
4. Evaporate the filtrate once more to a 
syrup. 
5. Add alcohol again, stirring until all is 
separated which is not soluble. 
6. Filter once more, drive off the alcohol by 
evaporation, dissolve the residue in water, 
render alkaline by NaOH, and shake in a sepa- 
rating funnel with ether. Strychnine, vera- 
trine, atropine, and cocaine are found in the 
ether, which is allowed to evaporate spontane- 
ously in an evaporating dish, and the residue 
verified by the above tests. 
7. Free from ether by warming upon the 
water-bath, add ammonium chloride until am- 
moniacal, and allow to stand over night. Then 
warm, and shake with amyl alcohol. Morphine 
found in the amyl alcohol. Allow to evaporate, 
and verify by the above tests. 82 
A Laboratory Manual 
V. 
Scheme for the Detection of 
an Unknown Poison. 
A fresh specimen is to be taken for each test. 
1. Add methyl-violet or dimethyl-amido- 
azo-benzol. 
Shows free mineral acids : 
Sulphuric. 
Nitric. 
Hydrochloric (volatile). 
2. Add ferric chloride. Blue color shows 
carbolic acid (volatile). 
3. Take a portion in 3 test-tubes and acidify 
with HO. Place in : 
(a.) A piece of bright copper tinsel, and 
warm. Deposit shows arsenic, mercury, or 
antimony. 
(b.) A piece of bright tin. Black deposit 
in the cold shows antimony. 
(c.) A piece of bright steel. Red deposit 
shows copper. 
4. Add potassium iodide. Yellow pp. Or 
moisten a piece of filter-paper with the solu- Of Physiological Chemistry 
83 
tion and place in a stream of II2S. Black 
shining deposit shows 
Lead. 
lake a portion in 2 test-tubes. Place 
in : 
(ci.) A strip of filter-paper moistened with 
guaiac mixture. Warm gently. Blue color 
shows hydrocyanic acid. 
(b.) A strip of filter-paper moistened with 
silver nitrate. TV arm gently. Black deposit 
in the dark shows phosphorus. 
6. Distil into water. In distillate is found: 
Hydrochloric acid. 
Carbolic acid. 
Hydrocyanic acid. 
Phosphorus. 
Nitro-benzol. 
7. Extract with alcohol: 
Sulphuric acid. 
Nitric acid. 
Oxalic acid. 
8. Extract with alcohol and tartaric acid 
(Stas-Otto) : 
Morphine. 
Strychnine. 
Atropine. 
Veratrine. 
Cocaine. 84 
A Laboratory Manual 
9* Destroy organic matter with hydrochloric 
acid and potassium chlorate (Fresenius-Babo) : 
Arsenic. 
Antimony. 
Mercury. 
Lead. 
Copper. APPENDIX. Reagents. 
Alcohol. —The alcohol must be colorless 
and leave no residue upon evaporation. 
Sulphuric Ether. 
Ammonia. — Liq. ammon. caust. 
Ammonium Carbonate. 
Alkaline Barium Solution (Baryta mixt- 
ure) . — Equal parts barium water and barium 
chloride solution. 
Barium Water. — 1 part caustic barium. 
* 15 parts water. 
Dissolved warm, cooled, and filtered. 
Barium Nitrate. — 1 : 12. 
Barium Chloride. — 1: 10. 
Barium Carbonate. — Precipitated out of a 
barium chloride solution by ammonium car- 
bonate and well washed. 
Lead Acetate. — 1 : 10. 
Subacetate of Lead Solution. — (Liq. plumbi 
subacetatis U.S.P.) 
Bromine Water.— Water treated with an 
excess of bromine and cleared by allowing to 
stand, and decanting. 
Ammonium Chloride. — 1 : 10. 
Calcium Chloride. — 1 : 10. 
87 88 
A Laboratory Manual 
Sodium Chloride. — Cold saturated solution. 
Alcoholic Zinc Chloride. — Thick syrupy 
watery solution diluted with alcohol until the 
spec. grav. is 1.2. 
Cochineal Tincture. — 5 grammes cochineal 
with 150 c.c. alcohol and 100 c.c. water. 
Allow to stand some days at room temperature, 
pour off, and filter. 
Acetic Acid. — 30 % • 
Ferric Chloride. — 3 : 100. 
Potassium Ferrocyanide. — 1 : 10. 
Yellow Chromate of Potash (K2Cr04).— 
1 : 20. 
Potassium Nitrate. — Must be free from 
chlorides. 
Potassium Hydrate. — 1 part potassium hy- 
drate. 
2 parts water. 
Copper Sulphate. — 1 : 10. 
Magnesium Sulphate. — Cold saturated solu- 
tion. 
Magnesium Mixture. — 1 part crystallized 
MgS04. 
2 parts ammonium 
chloride. 
4 parts ammonia. 
8 parts water. 
Millon’s Reagent. — 1 part mercury with 2 Of Physiological Chemistry 
89 
parts nitric acid of spec. gray. 1.4. Gently 
warmed until the mercury is fully dissolved. 
One part of this solution is treated with 2 parts 
water. 
Ammonium Molybdate. — 50 grammes of 
ammonium molybdate are dissolved in 200 
grammes of ammonia. The solution is poured 
into 750 c.c. nitric acid of spec. grav. 1.2; 
allowed to stand some days in a warm place, 
and decanted. 
Sodium Hydrate. — Spec. grav. 1.17. 
Sodium Carbonate. — Saturated solution. 
Sodium Phosphate (Na2HP04).— 1: 10. 
Nessler’s Reagent. 50 grammes potassium 
iodide dissolved in an equal amount of water, 
and as much mercuric chloride added while 
warming until the mercury is no longer dis- 
solved. Then add a solution of 150 grammes 
KOH in 300 c.c. water, fill to a liter, add 5 c.c. 
more mercuric chloride solution, and let stand. 
Nylander’s Solution. — 4 grammes tartarate 
of sodium and po- 
tassium. 
2 grammes bismuth 
subnitrate. 
10 grammes NaOH 
in 90 c.c. water. 
To be kept in a dark bottle. 90 
A Laboratory Manual 
Ammonium Oxalate. — 1 : 25. 
Mercuric Chloride. — 1:20. 
Phospho-Tungstic Acid. — 1 : 20, acidified 
with HC1. 
Potassium Sulphoeyanide. — 1:20. 
Rosolic Acid. — 1 : 100 alcohol. 
Hydrochloric Acid. — 1.124 spec. gray. 
Nitric Acid. — Must be colorless ; free from 
nitrous acid and HC1. Spec. gray. 1.2. 
Sulphuric Acid. — 200 grammes cone. H2S04 
to 1 liter. 
Silver Nitrate. — 1:30. 
Tartaric Acid. — Crystals or powder. 
Lactic Acid Solution. — 0.8 gramme : 100 
c.c. water. 
Digestive HC1 Solution. — 10 c.c. 25% HC1 
to 1 liter water. 
Sol. Methyl-Violet. — 0.5:1,000. 
Sol. Tropaeolin OO. — 0.25:1,000. 
Giinzburg’s Reagent.— 1 gramme vanillin. 
2 grammes phloro- 
glucin. 
100 c.c. alcohol. 
Artificial Gastric Juice.—0.5 gramme pep- 
sin dissolved in 500 c.c. digestive HC1 solu- 
tion . Of Physiological Chemistry 
91 
Frolule’s Reagent.— 0.1 gramme sodium 
molybdate dissolved in 10 c.c. cone. II2S04. 
Pacini’s Fluid. — Mercuric chloride, 2 
grammes. 
Sodium chloride, 4 grammes. 
Glycerine, 26 grammes. 
Distilled water, 226 grammes. 
Guaiac Mixture. — 0.5 gramme gum guaiac 
dissolved in 10 c.c. alcohol, to which are added 
a few drops weak copper sulphate solution 
(1 : 2,000). The mixture must be kept tightly 
corked. 
Saltpeter Mixture. — 3 parts KN03. 
1 part Na2C03. 
Dimetliyl-amido-azo-benzol. — 0.5 gramme 
to 100 c.c. alcohol. 
Sub-cliloride of Tin. — Metallic tin dissolved 
in excess in HC1. 
Tannic Acid. — 1 : 100. 
Indigo Carmine. — Solution. 
Alpha-lSTaphthol. —15% solution in alcohol. 
Iodine in Potassium Iodide (IKI sol.). — 
Iodine, 1 gramme. 
Potassium iodide, 2 grammes. 
Water, 300 c.c. 
Picric Acid. — Saturated solution. 92 
A Laboratory Manual 
Sodium Acetate Solution. — 100 grammes 
acetate of sodium dissolved in distilled water, 
and 100 c.c. of a 30% solution of acetic acid, 
the whole being diluted to a liter. 
Sulphanilic Acid. — Sulphanilic acid, 2 
grammes. 
HC1, 50 c.c. 
Distilled water, 1,000 c.c. 
Diazo solution No. 1. 
Sodium Nitrite. — 0.5% solution. 
Diazo solution No. 2. 
Standard Solutions. 
N Solution Tartaric Acid (for alkalinity 
of blood). — Contains 3 grammes tar- 
taric acid to 1 liter distilled water. 
1 c.c. = .0016 gramme NaOH. 
Standardized with a — solution NaOH. 
10 
N Solution NaOH (for acidity of gastric 
juice). — Contains 4 grammes NaOH 
to 1 liter distilled water. 
1 c.c. = .00365 gramme HC1, 
Standardized with — solution H9SO,,. 
10 2 Of Physiological Chemistry 
93 
Esbach’s Reagent (for quantitative albu- 
men). — 10 grammes picric acid. 
20 grammes citric acid. 
Dissolved in 1,000 c.c. distilled water. 
Fehling’s Solution (for quantitative sugar). 
— 34.64 grammes of pure crystallized copper 
sulphate, dissolved in about 200 c.c. distilled 
water. 
173 grammes potassium and sodium tar- 
tarate. 
125 grammes NaOH. 
Both dissolved in 500 c.c. distilled water. 
These two solutions are mixed together, and 
diluted with distilled water to 1 liter. 
10 c.c. = .050 gramme glucose. 
Standardized with a solution of glucose, 5 
grammes to 1 liter distilled water. 
© 
Standard Solution Mercuric Nitrate (for 
quantitative urea).— 71.48 grammes pure 
metallic mercury, dissolved with warming in 
pure cone. HN03, and diluted to 1 liter with 
distilled water. 
1 c.c. = .010 gramme urea. 
Standardized with a solution of urea, 2 
grammes to 100 c.c. distilled water. A Laboratory Manual 
94 
jN Solution Potassium Permanganate (for 
520 .... v 
quantitative uric acid). — 1.578 grammes 
pure crystals potassium permanganate to 1 
liter distilled water. 
1 c.c. =3.61 milligrammes uric acid. 
N 
Standardized with — solution oxalic acid. 
20 
Standard Solution Silv er Nitrate (for quan- 
titative chlorides). — 29.06 grammes pure sil- 
ver nitrate to 1 liter distilled water. 
1 c.c. = .010 gramme Nad. 
Standardized with a solution Nad, 10 
grammes to 1 liter distilled water. 
Standard Solution Uranyl Nitrate (for quan- 
titative phosphoric acid). — 33 grammes ura- 
nium oxide dissolved in pure HN03 and diluted 
to 1 liter. 
1 c.c. = .005 gramme P205. 
Standardized with 10.085 grammes Na2HP04 
to 1 liter distilled water. Of Physiological Chemistry 
95 
Analysis of Blood. 
Date. Name. 
Qualitative Tests. 
Oxy haemoglobin. Methaemoglobin. 
Carbon-monoxide-hae- Haematin. 
moglobin. Haemin crystals. 
Sulpho-haemoglobin. 
Quantitative Tests. 
Specific gravity. Alkalinity. 
Haemoglobin. (Fleischl’s Haemometer.) 
Number of red corpuscles. (By Thoma-Zeiss 
method and Haematokrit.) 
Number of white corpuscles. 
Relation of white to red. 
Diagnosis. 
Remarks. 96 
A Laboratory Manual 
Analysis of Gastric Contents. 
Date. Name. 
Period after test meal. 
Qualitative Tests. 
Free HC1. Pepsin. Acetic acid. 
Lactic acid. Amylaceous matter. Butyric acid. 
Rennet. Albumose-peptone. Formic acid. 
Quantitative Tests. 
Total acidity. 
Total HC1 (free and combined). 
Microscopic Analysis. 
Sarcime. 
Yeast fungi. 
Diagnosis. 
Remarks.